This program continues to study the tissue specific and developmental regulation of the osteocalcin (OC) gene and the factors that contribute to induction and progression of osteoblast (OB) maturation. In the current program period, we discovered a regulatory network of homeodomain (HD) proteins that provide molecular switches for mediating the temporal expression of OC with a pivotal activating role for Dlx3 at the onset of OC transcription. A novel regulatory element for the Hox transcription factors was discovered that binds Hoxa10 for activation of OC transcription. Hoxa10 and Dlx3 are bound to the OC gene prior to Runx2 and increase again with Runx2 binding. In addition, these factors can activate the OC gene in Runx2 null cells, thus providing evidence for molecular events contributing to expression of the OC and bone related genes upstream of Runx2. These findings justify further investigation of their functional roles in osteoblasts in vivo and in vitro. We postulate that these two regulatory factors have key roles in regulating gene expression throughout OB differentiation, a function distinct from their activities in embryonic patterning of the skeleton. We will identify these OB- related functions by determining the mechanism by which they regulate developmental expression of OC which include contributing to chromatin remodeling, regulating activation and repression of gene transcription through interactions with co-regulatory factors, and functioning both independent of Runx2 and coordinated with Runx2 for establishment of the OB phenotype. Aim1 tests the hypothesis that dynamic association/dissociation of these factors with osteocalcin chromatin occurs at specific stages of phenotype development due to formation of transcription factor complexes modifying chromatin architecture of OC. Aim 2 addresses the hypothesis that Hoxa10 functions upstream of Runx2 to promote epigenetic marking of the OC gene for tissue specific activation, and regulation of target gene expression in OBs. Its deletion from OB in vitro and in vivo is postulated to alter OB maturation and contribute to a mild bone phenotype in adult mice. Aim 3 postulates that Dlx3 has a transient role in gene regulation, responsible for the timing of transcription of the OC gene and coordinated occupancy of Runx2 which will be addressed ex vivo in cells from the Dlx3 null and conditional knockout mice. We further postulate that the human Dlx3 tricho-dento-osseous (TDO) mutation alters the transient function of Dlx3 in osteoblasts as a result of loss-of-function of interacting co-regulatory proteins. These studies will identify novel mechanisms and regulatory pathways required for induction and regulation of OC and regulation of OB maturation to the differentiated phenotype in a mineralizing matrix.